Effect Of Phosphorylation Status Of Receptor Cytoplasmic Kinas E Strk1 On Its Function In Rice

Rice(Oryza sativa L.)is one of the most important and high-yield food crops in China and even in the world.Rice has raised more than half of the world's population.Salt stress seriously limits the growth of rice and is one of the major adverse factors restricting the development of modern agriculture.Previous studies found that STRK1(salt tolerance receiver-like cytoplasmic kinase 1)can phosphorylate CatC at Tyr210 and activate its activity to regulate the homeostasis of reactive oxygen species(ROS)under salt stress,improving the salt tolerance of rice.However,the effect of phosphorylation stasus of STRK1 on its function during signal transmission is still unclear.In order to explore the mechanism,we examin the phosphorylation site of STRK1 that plays an important role in the process of signal transmission from STRK1 to CatC,which is important for us to understand the effect of phosphorylation state of STRK1 on its function.We carried out a series of experiments,and the main results are as follows:(1)We Purified STRK1 and CatC recombinant proteins from Ecoli,and carried out phosphorylation reactions in vitro.Then the Post-reaction proteins were subject to mass spectrometry analysis to identify their phosphorylated sites.The phosphorylated sites of STRK1 were identified,including 17 serine sites,3 tyrosine sites and 3 threonine sites.The 3 tyrosine phosphorylation sites(Y82,Y240,Y258)indicates that there is tyrosine cascade phosphorylation during the signal transmission.(2)In order to determine the interaction domian between STRK1 and CatC,CatC is divided into three segments,Respectively,N-terminal(CatC-NT),catalase domain(CatC-CD)and C-terminal(CatC-CT).It is found that STRK1 mainly interacts with CatC-CD segment through yeast two-hybrid assay.We also confirmed that the single phosphorylation site mutant of STRK1 and CatC had no effect on their interaction in yeast.(3)In order to explore the phosphorylation sites on STRK1 that play important roles in the signal transmission,we constructed the Tyrosine sites mutant STRK1,purified the mutant protein and played the phosphorylation reaction in vitro.Our results showed that mutation of 3 tyrosine phosphorylation sites of STRK1 will affect the ability of phosphorylation substrate CatC.Indicate the 3 tyrosine phosphorylation sites play a crucial part in signal transmission,but which is the major one to be further analysis.(4)In order to further verify the above results in vivo,it is necessary to obtain a STRK1 mutant that can be used for recovery.Therefore,we constructed the STRK1 knockout lines by CRISPR/Cas9 system knockout vector of STRK1 is constructed,and obtained 2 STRK1 knockout lines(strk1-1 and strk1-3).Phenotypic analysis under salt stress showed that the knockout lines reduce the salt tolerance of rice.After further isolation and identification,a knockout line containing no T-DNA insertion was identified,which can be used to further verify the effect of STRK1 phosphorylation status on its function in vivo.To sum up,the auto-phosphorylation sites on STRK1 was identified in vitro,and the interaction between catalytic domains of STRK1 and CatC was determined by yeast two-hybrid method.Furthermore,the mutation of three Tyrosine phosphorylation sites on STRK1 would weaken its ability to phosphorylate CatC,indicate that these 3 tyrosine phosphorylation sites play an improtant role in signal transmission.CRISPR/Cas9 knockout lines of STRK1 was successfully constructed and obtained.It Provides material basis to further verify the effect of STRK1 phosphorylation state on its function in vivo.